Comparative Analysis of Dehydrins from Woody Plant Species.

We conducted analyses on 253 protein sequences (Pfam00257) derived from 25 woody plant species, including trees, shrubs, and vines. Our goal was to gain insights into their architectural types, biochemical characteristics, and potential involvement in mitigating abiotic stresses, such as drought, cold, or salinity. The investigated protein sequences (253) comprised 221 angiosperms (85 trees/shrubs and 36 vines) and 32 gymnosperms. Our sequence analyses revealed the presence of seven architectural types: Kn, KnS, SKn, YnKn, YnSKn, FSKn, and FnKn. The FSKn type predominated in tree and shrub dehydrins of both gymnosperms and angiosperms, while the YnSKn type was more prevalent in vine dehydrins. The YnSKn and YnKn types were absent in gymnosperms. Gymnosperm dehydrins exhibited a shift towards more negative GRAVY scores and Fold Indexes. Additionally, they demonstrated a higher Lys content and lower His content. By analyzing promoter sequences in the angiosperm species, including trees, shrubs, and vines, we found that these dehydrins are induced by the ABA-dependent and light-responsive pathways. The presence of stress- and hormone-related cis-elements suggests a protective effect against dehydration, cold, or salinity. These findings could serve as a foundation for future studies on woody dehydrins, especially in the context of biotechnological applications.

The Precision Analysis of Cutting Edge Preparation on CBN Cutting Inserts Using Rotary Ultrasonic Machining.

This paper is focused on the issue of preparing the cutting edge microgeometry of cutting inserts made of cubic boron nitride (CBN). The aim of this research was to investigate the possibilities of rotary ultrasonic machining (RUM) for preparing asymmetric cutting edge microgeometries of various shapes (chamfers, circular, and elliptical rounding and their combinations) on a CBN cutting tool. In this article, a new type of advanced cutting edge preparation method is presented. CBN is relatively resistant to the most often used (abrasive) methods of cutting-edge preparation, due to its very high hardness (which is a prerequisite property for machining difficult-to-cut materials). Such hard materials could be processed using advanced manufacturing methods, and rotary ultrasonic machining (RUM) is one such method. Experiments have shown that RUM can be used for machining CBN. However, high hardness is not the only challenge here. For cutting edge preparation, it is necessary to achieve an adequate accuracy of size and dimensions. The presented paper analyzes the suitability of the RUM process for processing CBN inserts. The results of the experiment showed that this method can be used for preparing asymmetric cutting edge microgeometries with various shapes.

The humidity level matters during the desiccation of Norway spruce somatic embryos.

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes ß-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some ß-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

ß-1,3-Glucanases and chitinases participate in the stress-related defence mechanisms that are possibly connected with modulation of arabinogalactan proteins (AGP) required for the androgenesis initiation in rye (Secale cereale L.).

This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, ß-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of ß-1,3-glucanases and chitinases. Some isoforms of ß-1,3-glucanases, namely several acidic isoforms of about 26 kDa; appeared to be anther specific. Chitinases were well represented but were less variable. RT-qPCR revealed that the cold-responsive chitinase genes Chit1 and Chit2 were expressed at a lower level in the microspores and whole anthers while the cold-responsive Glu2 and Glu3 were not active. The stress pre-treatments modifications promoted the AGP accumulation. An apparent dominance of some AGP epitopes (LM2, JIM4 and JIM14) was detected in the androgenesis-responsive rye line. An abundant JIM13 epitopes in the vesicles and inner cell walls of the microspores and in the cell walls of the anther cell layers appeared to be the most specific for embryogenesis.

Diacylglycerol Acetyltransferase Gene Isolated from Euonymus europaeus L. Altered Lipid Metabolism in Transgenic Plant towards the Production of Acetylated Triacylglycerols.

Euonymus species from the Celastraceae family are considered as a source of unusual genes modifying the oil content and fatty acid composition of vegetable oils. Due to the possession of genes encoding enzyme diacylglycerol acetyltransferase (DAcT), Euonymus plants can synthesize and accumulate acetylated triacyglycerols. The gene from Euonymus europaeus (EeDAcT) encoding the DAcT was identified, isolated, characterized, and modified for cloning and genetic transformation of plants. This gene has a unique nucleotide sequence and amino acid composition, different from orthologous genes from other Euonymus species. Nucleotide sequence of original EeDAcT gene was modified, cloned into transformation vector, and introduced into tobacco plants. Overexpression of EeDAcT gene was confirmed, and transgenic host plants produced and accumulated acetylated triacylglycerols (TAGs) in immature seeds. Individual transgenic plants showed difference in amounts of synthesized acetylTAGs and also in fatty acid composition of acetylTAGs.

Experimental Study of Drilling Temperature, Geometrical Errors and Thermal Expansion of Drill on Hole Accuracy When Drilling CFRP/Ti Alloy Stacks.

The drilling of holes in CFRP/Ti (Carbon Fiber-Reinforced Plastic/Titanium alloy) alloy stacks is one of the frequently used mechanical operations during the manufacturing of fastening assemblies in temporary civil aircraft. A combination of inhomogeneous behavior and poor machinability of CFRP/Ti alloy stacks in one short drilling brought challenges to the manufacturing community. The impact of the drilling temperature and time delay factor under various cutting conditions on hole accuracy when machining CFRP/Ti alloy stacks is poorly studied. In this paper, the drilling temperature, the phenomenon of thermal expansion of the drill tool, and hole accuracy are investigated. An experimental study was carried out using thermocouples, the coordinate measuring machine method, and finite element analysis. The results showed that the time delay factor varied from 5 (s) to 120 (s), influences the thermal-dependent properties of CFRP, and leads to an increase in hole roundness. Additionally, the thermal expansion of the drill significantly contributes to the deviation of the hole diameter in Ti alloy.

Expression of Drosera rotundifolia Chitinase in Transgenic Tobacco Plants Enhanced Their Antifungal Potential.

In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin. The protein extracts of these two lines exhibited five and eight times higher chitinolytic activity than non-transgenic controls when measured in a fluorimetric assay with FITC-chitin. Finally, the growth of Trichoderma viride was obviously suppressed when the pathogen was exposed to 100 µg of crude protein extract isolated from line 9 and line 14, with the area of mycelium growth reaching only 56.4% and 45.2%, of non-transgenic control, respectively. This is the first time a chitinase from a carnivorous plant with substrate specificity for long chitin polymers was tested in a transgenic plant with the aim of exploring its antifungal potential.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

The pollen- and embryo-specific Arabidopsis DLL promoter bears good potential for application in marker-free Cre/loxP self-excision strategy.

KEY MESSAGE: Marker-free transgenic plants can be generated with high efficiency by using the Cre/ lox P self-excision system controlled by the pollen- and embryo-specific Arabidopsis DLL promoter. In this work, we aimed to study the feasibility of using the pollen- and embryo-specific DLL promoter of the At4g16160 gene from Arabidopsis thaliana in a Cre/loxP self-excision strategy. A Cre/loxP self-excision cassette controlled by the DLL promoter was introduced into the tobacco genome via Agrobacterium-mediated transformation. No evidence for premature activation of the Cre/loxP system was observed in primary transformants. The efficiency of nptII removal during pollen and embryo development was investigated in transgenic T1 progenies derived from eight self- and four cross-pollinated T0 lines, respectively. Segregation and rooting assays were performed to select recombined T1 plants. Molecular analyses of these plants confirmed the excision event in all analysed T0 lines and marker-free transgenic T1 plants were obtained with efficiency of up to 96.2%. The Arabidopsis DLL promoter appears to be a strong candidate to drive Cre-mediated recombination not only in tobacco as a model plant, but also in other plant species.

Plant chitinase responses to different metal-type stresses reveal specificity.

KEY MESSAGE: Chitinases in Glycine max roots specifically respond to different metal types and reveal a polymorphism that coincides with sensitivity to metal toxicity. Plants evolved various defense mechanisms to cope with metal toxicity. Chitinases (EC 3.2.1.14), belonging to so-called pathogenesis-related proteins, act as possible second line defense compounds in plants exposed to metals. In this work their activity was studied and compared in two selected soybean (Glycine max L.) cultivars, the metal-tolerant cv. Chernyatka and the sensitive cv. Kyivska 98. Roots were exposed to different metal(loid)s such as cadmium, arsenic and aluminum that are expected to cause toxicity in different ways. For comparison, a non-metal, NaCl, was applied as well. The results showed that the sensitivity of roots to different stressors coincides with the responsiveness of chitinases in total protein extracts. Moreover, detailed analyses of acidic and neutral proteins identified one polymorphic chitinase isoform that distinguishes between the two cultivars studied. This isoform was stress responsive and thus could reflect the evolutionary adaptation of soybean to environmental cues. Activities of the individual chitinases were dependent on metal type as well as the cultivar pointing to their more complex role in plant defense during this type of stress.

The influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the "mild" heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 µm, endogenous auxin content in a single cell corresponded to concentration of 1.01 µM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

Glucan-rich diet is digested and taken up by the carnivorous sundew (Drosera rotundifolia L.): implication for a novel role of plant ß-1,3-glucanases.

Carnivory in plants evolved as an adaptation strategy to nutrient-poor environments. Thanks to specialized traps, carnivorous plants can gain nutrients from various heterotrophic sources such as small insects. Digestion in traps requires a coordinated action of several hydrolytic enzymes that break down complex substances into simple absorbable nutrients. Among these, several pathogenesis-related proteins including ß-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-ß-1,3-glucanase of ~50 kDa is present in the digestive fluid of the flypaper-trapped sundew (Drosera rotundifolia L.). The enzyme is inducible with a complex plant ß-glucan laminarin from which it releases simple saccharides when supplied to leaves as a substrate. Moreover, thin-layer chromatography of digestive exudates showed that the simplest degradation products (especially glucose) are taken up by the leaves. These results for the first time point on involvement of ß-1,3-glucanases in digestion of carnivorous plants and demonstrate the uptake of saccharide-based compounds by traps. Such a strategy could enable the plant to utilize other types of nutritional sources e.g., pollen grains, fungal spores or detritus from environment. Possible multiple roles of ß-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

Cultivar-specific kinetics of chitinase induction in soybean roots during exposure to arsenic.

The kinetics of defense responses was studied in soybean exposed to ecologically relevant concentrations of arsenic for 96 h. In the roots of two soybean cultivars with contrasting tolerance to this metalloid there were observed differences in basal levels of membrane lipid peroxidation as well as a significantly different course of peroxidation upon exposure to As. The different course of stress was reflected in the accumulation of defense components. The responses of individual chitinase isoforms were studied since these enzymes had previously been shown to be stable components of defense against metals. The kinetics and magnitude of accumulation of the three isoforms during exposure to As significantly differed within as well as between the studied cultivars. Furthermore, accumulation of these isoforms appeared to be related to oxidative status in the root tissue. The timing of induced responses is likely to be important for efficient defense against metal(oid) pollution in environment.

Defense responses of soybean roots during exposure to cadmium, excess of nitrogen supply and combinations of these stressors.

Heavy metal pollution is a serious environmental problem in agricultural soils since the uptake of heavy metals by plants represents an entry point into the food chain and is influenced by the form and amount of nitrogen (N) fertilization. Here we studied the defense responses in soybean roots exposed to ions of cadmium (applied as 50 mg l(-1) Cd(2+)) when combined with an excessive dose of N in form of NH(4)NO(3). Our data indicate that despite of stunted root growth, several stress symptoms typically observed upon cadmium treatment, e.g. peroxidation of lipid membranes or activation of chitinase isoforms, become suppressed at highly excessive N. At the same time, other defense mechanisms such as catalases and proline accumulation were elevated. Most importantly, the interplay of ongoing responses resulted in a decreased uptake of the metal into the root tissue. This report points to the complexity of plant defense responses under conditions of heavy metal pollution combined with intensive fertilization in agriculture.

Biochemical and physiological comparison of heavy metal-triggered defense responses in the monocot maize and dicot soybean roots.

Defense responses against cadmium, arsenic and lead were compared in two crop plants such as the monocotyledonous maize (Zea mays cv. Quintal) and dicotyledonous soybean (Glycine max cv. Korada). The applied metals caused root growth retardation, membrane damage and subsequent loss of cell viability, while enhanced H(2)O(2) generation, lipid peroxidation and lignification were detected with respect to corresponding controls. The measured data suggest that soybean was in general more tolerant to tested doses of metals and showed more pronounced defense responses than maize. Concurrently, the total activity of ß-1,3-glucanases, a subgroup of so called pathogenesis-related defense proteins, was comparable in root extracts of both plant types. Though in a view of previous comparative genome approaches the ß-1,3-glucanases do not mirror the differences in the cell wall structure and architecture between the monocots and dicots, we show that in both plant types they clearly respond to metal stress. Accumulation patterns of different glucanase isoforms upon exposure to tested metals indicate that they do contribute to plant defense mechanisms during exposure to heavy metals and their biological role is more complex than expected.

Agrobacterium-mediated transformation of embryogenic tissues of hybrid firs (Abies spp.) and regeneration of transgenic emblings.

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba x A. cephalonica (cell lines AC2, AC78) and Abies alba x A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l(-1)). The T-DNA of plant transformation vector contained the beta-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l(-1). GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.

Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated.

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants.

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T(0) plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T(0) transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T(0)-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T(2) seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T(1) plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.